Celecoxib analogs possessing a N-(4-nitrooxybutyl)piperidin-4-yl or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-yl nitric oxide donor moiety: Synthesis, biological evaluation and nitric oxide release studies

Article abstract of DOI:10.1016/j.bmcl.2010.01.014

A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) coxib prodrugs (NO-coxibs) wherein the para-tolyl moiety present in celecoxib was replaced by a N-(4-nitrooxybutyl)piperidyl 15a-b, or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl 17a-b, NO-donor moiety was synthesized. All compounds released a low amount of NO upon incubation with phosphate buffered saline (PBS) at pH 7.4 (2.4-5.8% range). In comparison, the percentage NO released was higher (3.1-8.4% range) when these nitrate prodrugs were incubated in the presence of l-cysteine. In vitro COX-1/COX-2 isozyme inhibition studies showed this group of compounds are moderately more potent, and hence selective, inhibitors of the COX-2 relative to the COX-1 enzyme. AI structure-activity relationship data acquired showed that compounds having a MeSO₂ COX-2 pharmacophore exhibited superior AI activity compared to analogs having a H₂NSO₂ substituent. Compounds having a MeSO₂ COX-2 pharmacophore in conjunction with a N-(4-nitrooxybutyl)piperidyl (ED₅₀ = 132.4 mg/kg po), or a N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl (ED₅₀ = 118.4 mg/kg po), moiety exhibited an AI potency profile that is similar to aspirin (ED₅₀ = 128.7 mg/kg po) but lower than ibuprofen (ED₅₀ = 67.4 mg/kg po).

Full text of DOI:10.1016/j.bmcl.2010.01.014

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1324–1329
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Celecoxib analogs possessing a N-(4-nitrooxybutyl)piperidin-4-yl
or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-yl nitric oxide donor moiety:
Synthesis, biological evaluation and nitric oxide release studies
Morshed A. Chowdhury, Khaled R. A. Abdellatif, Ying Dong, Gang Yu, Zhangjian Huang, Moshfiqur Rahman,
*
Dipankar Das, Carlos A. Velázquez, Mavanur R. Suresh, Edward E. Knaus
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alta, Canada T6G 2N8
a r t i c l e i n f o
a b s t r a c t
Article history:
A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) coxib prodrugs (NO-coxibs)
wherein the para-tolyl moiety present in celecoxib was replaced by a N-(4-nitrooxybutyl)piperidyl
15a–b, or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl 17a–b, NO-donor moiety was synthesized. All
compounds released a low amount of NO upon incubation with phosphate buffered saline (PBS) at pH
7.4 (2.4–5.8% range). In comparison, the percentage NO released was higher (3.1–8.4% range) when these
Received 18 December 2009
Revised 4 January 2010
Accepted 5 January 2010
Available online 11 January 2010
nitrate prodrugs were incubated in the presence of L-cysteine. In vitro COX-1/COX-2 isozyme inhibition
Keywords:
Piperidine
studies showed this group of compounds are moderately more potent, and hence selective, inhibitors of
the COX-2 relative to the COX-1 enzyme. AI structure–activity relationship data acquired showed that
compounds having a MeSO2 COX-2 pharmacophore exhibited superior AI activity compared to analogs
having a H2NSO2 substituent. Compounds having a MeSO2 COX-2 pharmacophore in conjunction with
a N-(4-nitrooxybutyl)piperidyl (ED50 = 132.4 mg/kg po), or a N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyr-
idyl (ED50 = 118.4 mg/kg po), moiety exhibited an AI potency profile that is similar to aspirin
(ED50 = 128.7 mg/kg po) but lower than ibuprofen (ED50 = 67.4 mg/kg po).
1,2,3,6-Tetrahydropyridine
Nitrate prodrugs
Cyclooxygenase-1 and cyclooxygenase-2
inhibition
Anti-inflammatory activity
Ó 2010 Elsevier Ltd. All rights reserved.
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely
used for the treatment of pain, fever, and inflammation. The use
of NSAIDs that selectively inhibit the inducible cyclooxygenase-2
(COX-2) isozyme in the periphery provided a useful drug design
concept. This discovery resulted in the development of effective
anti-inflammatory (AI) drugs that were devoid of adverse cardio-
vascular effects and gastrointestinal ulcerogenicity believed to be
associated with inhibition of the constitutive cyclooxygenase iso-
form (COX-1).1 Therefore, COX-2 selective inhibitors (coxibs) such
as celecoxib (CelebrexÒ, 1), rofecoxib (VioxxÒ, 2), and valdecoxib
(BextraÒ, 3a) were developed for the long term treatment of pa-
tients suffering from chronic pain and inflammation.2 Unfortu-
nately, some selective COX-2 inhibitory drugs that include
rofecoxib 2 and valdecoxib 3a alter the natural balance in the
COX biochemical pathway (see structures in Fig. 1). In this regard,
the amount of the desirable vasodilatory and anti-aggregatory
prostacyclin (PGI2) produced is decreased together with a simulta-
neous increase in the level of the undesirable prothrombotic
thromboxane A2 (TxA2).3–5 These two adverse biochemical changes
in the COX pathway are believed to be responsible for increased
incidences of high blood pressure and myocardial infarction that
ultimately prompted the withdrawal of rofecoxib (VioxxÒ) and val-
decoxib (BextraÒ).6,7
Nitric oxide (NO) exhibits a number of useful pharmacological
actions that include vascular relaxation (vasodilation), and inhibi-
tion of platelet aggregation and adhesion.8 Hybrid COX-2 inhibitors
possessing a NO-donor moiety (NO-coxibs) have been investigated
as a method to increase the clinical safety of COX-2 inhibitors. In
this regard, NO-coxibs having a nitrate ester NO-donor moiety
such as the oxazole (3b) exhibit anti-inflammatory activity similar
to valdecoxib with antithrombotic action at higher doses,9 and the
pyrazole 4 that exhibits anti-inflammatory activity.10 Accordingly,
we showed that a novel group of hybrid NO-releasing NONO-
NSAID ester prodrugs possessing a 1,3-dinitrooxy-2-propyl moiety
attached directly to the carboxylic group of aspirin (6b), indometh-
acin (7b) or ibuprofen (9b) showed approximately equipotent anti-
inflammatory activity to the parent NSAID, without significant
gastric toxicity when administered orally.11 (see structures in
Fig. 1). Studies carried out by others have shown that nitrate-based
NO-NSAIDs such as NO-aspirin (5, 6a),12,13 NO-indomethacin (7a,
8),14,15 and NO-ibuprofen (9a),15 are gastrointestinal sparing while
simultaneously suppressing prostaglandin synthesis similar to the
parent drugs. As part of our ongoing research program to develop
* Corresponding author. Tel.: +1 780 492 5993; fax: +1 780 492 1217.
E-mail address: eknaus@pharmacy.ualberta.ca (E.E. Knaus).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.014

M. A. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1324–1329
1325
Me
SO2NH2
SO2Me
SO2NH2
SO2NH2
N
O
N
N
O
O
R1
N
N
O2NO
F3C
Celecoxib (1)
Valdecoxib (3a, R1 = Me)
Rofecoxib (2)
NMI-1093 (3b, R1 = CH2ONO2)
Cl
O
N
6a, 7a; R1
6b, 7b; R1
=
=
ONO2
O
CH3
OAc
OR1
O
OR1
ONO2
MeO
ONO2
ONO2
O
O
OAc
7a (NO-Indomethacin)
7b (NONO-Indomethacin)
5 (NCX 4016, NO-Aspirin)
6a (NCX 4215, NO-Asprin)
6b (NONO-Aspirin)
Cl
O
N
OH
9a; R1
9b; R1
=
=
CH3
OR1
ONO2
CH3
OH
MeO
O
ONO2
ONO2
ONO2
O
O
i-Bu
9a (NO-Ibuprofen)
9b (NONO-Ibuprofen)
8 (NO-Indomethacin)
Figure 1. Chemical structures of the selective cyclooxygenase-2 (COX-2) inhibitors celecoxib (1), rofecoxib (2), and valdecoxib (3a), and the nitric oxide donor nitrate esters
NMI-1093 (3b) and 4, the 3-(nitrooxymethylphenyl) ester of aspirin (5), some nitrooxybutyl ester prodrugs of aspirin (6a) and indomethacin (7a), and the nitroglyceryl esters
of indomethacin (8) and ibuprofen (9a), and 1,3-dinitrooxy-2-propyl esters of aspirin (6b), indomethacin (7b), and ibuprofen (9b).
anti-inflammatory agents devoid of side effects, we now report the
synthesis, in vitro COX-1/COX-2 inhibitory activity, in vivo anti-
inflammatory activity and nitric oxide release data for a group of
coxib prodrugs that possess a N-(4-nitrooxybutyl)piperidyl 15a–
b, or a N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl 17a–b, NO-
donor moiety in place of the tolyl substituent present in celecoxib.
A group of 1-(4-methane(amino)sulfonylphenyl)-5-[1-(4-nitro-
oxybutyl)piperidin-4-yl]-3-trifluoromethyl-1H-pyrazoles (15a–b),
and 1-(4-methane(amino)sulfonylphenyl)-5-[1-(4-nitrooxybutyl)-
1,2,3,6-tetrahydropyridin-4-yl]-3-trifluoromethyl-1H-pyrazoles
(17a–b), hybrid nitric oxide donor prodrugs were synthesized
using the reaction sequence illustrated in Schemes 1 and 2, respec-
tively. Hydrogenation of 1-(4-methane(amino)sulfonylphenyl)-5-
(pyridin-4-yl)-3-trifluoromethyl-1H-pyrazole (10a–b) in glacial
acetic acid in the presence of Pd/C catalyst16 reduced the pyridine
ring to a piperidine ring to furnish the respective piperidyl prod-
ucts 11a–b in 70–77% yields. The observation that the C-4 piperi-
dine proton possessed two large vicinal coupling constants
(J3ax,4ax and J4ax,5ax = 12.2 Hz) indicates that the pyrazole ring in
11a–b assumes an equatorial orientation. An initial study was
directed toward synthesis of the target hybrid nitric oxide donor
N-diazen-1-ium-1,2-diolate derivative 13 of 1-(4-methanesulfo-
nylphenyl)-5-(piperidin-4-yl)-3-trifluoromethyl-1H-pyrazole (11a).
However, reaction of the piperidine compound 11a with nitric
oxide gas (40 psi) under a variety of experimental conditions
(aprotic and protic solvent systems, various temperatures) affor-
ded the N-nitrosopiperidine 12 as the sole isolable product rather
than the desired product 13 (see Scheme 1). For example, reaction
in the aprotic THF solvent system in the presence of NaOMe (95%)
at À65 °C afforded the N-nitrosopiperidyl product 12 in 67% yield.
Alternatively, reaction of 11a in a protic solvent system consisting
of NaOMe in MeOH (25% w/v) and MeCN at 25 °C furnished 12
(98%). The formation of the N-nitroso product 12 upon reaction
of 11a with nitric oxide indicates that the unstable intermediate
N-amino-N-diazen-1-ium-1,2-diolate product 13 must undergo
protonation of the diazen-1-ium-1,2-diolate N2-nitrogen which
subsequently eliminates a HNO species (see mechanism shown
in Scheme 1).17 On the other hand, reaction of the piperidyl
compounds 11a or 11b with 4-nitrooxybutyl bromide (14) in the
presence of cesium carbonate in dry DMF afforded the target N-
(4-nitrooxybutyl)piperidyl products 15a–b in 11–23% yield
(Scheme 1).
Alternatively, reaction of a 1,2,3,6-tetrahydropyridinyl com-
pound 16a or 16b with 14 furnished the respective N-(4-nitro-
oxybutyl)-1,2,3,6-tetrahydropyridyl product 17a or 17b in 27–
47% yields as illustrated in Scheme 2.
Hybrid nitric oxide donor prodrugs such as the N-(4-nitro-
oxybutyl)piperidines 15a–b and the corresponding 1,2,3,6-tetrahy-
dropyridine analogs 17a–b constitute a potential class of selective
COX-2 inhibitor agents that are devoid of adverse cardiovascular
properties. Compounds 15a–b and 17a–b were designed based
on structure–activity data showing that (i) a COX-2 pharmaco-
phore such as MeSO2 or H2NSO2 at the para-position of a N1-phenyl
ring on a pyrazole ring template confers potent and selective
COX-2 inhibitory activity,18 (ii) attachment of a 1,2,3,6-tetrahydro-
pyridine ring substituent in compounds 17a–b via their C-4 sp2

1326
M. A. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1324–1329
CF3
Hax
N
N
N
N
N
O
Na O
SO2Me
13
b
CF3
CF3
CF3
Hax
N
N
N
Hax
a
c
N
N
N
N
N
N
H
SO2R1
15a, R1 = Me
15b, R1 = NH2
SO2R1
10a, R1 = Me
10b, R1 = NH2
O2NO
SO2R1
11a, R1 = Me
11b, R1 = NH2
b
CF3
CF3
Hax
Hax
N
N
N
N
- HNO
N
O
H
N
N
N
N
O
+
O
SO2Me
12 (67-98%)
SO2Me
Scheme 1. Reagents and conditions: (a) AcOH, 10% Pd/C, H2 (60 psi), 25 °C, 20 h; (b) THF, NaOMe (95%), NO (40 psi), À65 °C, 1 h or MeCN, NaOMe (25% in MeOH), NO (40 psi)
25 °C, 24 h; (c) 4-nitrooxybutyl bromide (14), CsCO3, DMF, 25 °C, 16 h.
CF3
hybridized carbon atom is consistent with the observation that
two aryl rings on adjacent positions of a five-membered heterocy-
clic ring template (scaffold) generally provide optimum COX-2
inhibitory activity,19 (iii) the piperidyl and 1,2,3,6-tetrahydropyr-
idyl secondary amino group, such as that present in compounds
11 and 16, provides a logical synthon for elaboration to the corre-
sponding nitric oxide donor N-diazen-1-ium-1,2-diolates or N-ni-
trates,20–25 and (iv) the nitrooxybutyl group present in
compounds 15a–b and 17a–b could release a COX-2 inhibitory
compound and nitric oxide (NO).10 Unfortunately, reaction of the
piperidyl compound 11a with nitric oxide (see Scheme 1) afforded
the undesired N-nitroso product 12 rather than the desired unisol-
able N-diazen-1-ium-1,2-diolate product 13.
ONO2
N
N
HN
+
Br
14
SO2R1
16a, R1 = Me
16b, R1 = NH2
CsCO3, DMF
25 °C, 16 h
In vitro COX-1/COX-2 enzyme inhibition studies (Table 1)
showed that the piperidyl compound 15a did not inhibit the
COX-1 isozyme at the highest test compound concentration used
CF3
N
(100
compounds 15b and 17a–b were weak inhibitors of COX-1
(IC50 = 16.2–34.7 M range). Compounds 15a–b and 17a–b were
moderately more potent inhibitors of COX-2 (IC50 = 7.5–16.8
lM). Like the reference drug celecoxib (COX-1 IC50 = 7.7 lM),
O2NO
N
N
l
l
M
range) than COX-1 that resulted in a modest selectivity for the
COX-2 isozyme with COX-2 selectivity indexes in the 1.2 to >6
range. The COX-2 selectivity indexes shown by compounds 15a–
b and 17a–b are higher than reference drug aspirin (selectivity
index = 0.13) and comparable with ibuprofen (selectivity in-
dex = 2.64). The MeSO2 and H2NSO2 pharmacophores are not a
SO2R1
17a, R1 = Me
17b, R1 = NH2
Scheme 2.

M. A. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1324–1329
1327
Table 1
In vitro COX-1 and COX-2 inhibition, percent (%) nitric oxide release and anti-inflammatory (AI) data for 1-(4-methane(amino)sulfonylphenyl)-5-[1-(4-nitrooxybutyl)piperidin-4-
yl]-3-trifluoromethyl-1H-pyrazoles (15a–b) and 1-(4-methane(amino)sulfonylphenyl)-5-[1-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-yl]-3-trifluoromethyl-1H-pyrazoles
(17a–b), glycerol trinitrate (GTN, 18), and the reference drugs celecoxib, aspirin, and ibuprofen
CF3
CF3
Hax
N
N
N
N
N
N
CH2ONO2
CHONO2
CH2ONO2
O2NO
SO2R1
SO2R1
O2NO
17a, R1 = Me; 17b, R1 = NH2
15a, R1 = Me; 15b, R1 = NH2
18 (GTN)
COX-2 S.I.b
% NO releasedc
-Cysteinee
AI activityf: ED50 (mg/kg)
a
Compound
IC50
(lM)
COX-1
COX-2
PBSd
L
15a
15b
17a
17b
>100
19.8
34.7
16.2
—
7.7
0.3
2.9
15.6
16.8
12.8
7.5
>6
3.8
2.4
3.1
5.8
2.8
—
8.4
3.1
3.8
7.1
10.1
—
132.4 4.7
Weak activityg
118.4 4.1
Inactiveh
—
10.8
128.7
67.4
1.2
2.7
2.2
—
64.2
0.13
2.64
18
—
Celecoxib
Aspirin
Ibuprofen
0.12
2.4i
1.1i
—
—
—
—
a
The in vitro test compound concentration required to produce 50% inhibition of ovine COX-1 or human recombinant COX-2. The result (IC50, lM) is the mean of two
determinations acquired using the enzyme immuno assay kit (Catalog No. 560131, Cayman Chemicals Inc., Ann Arbor, MI, USA) and the deviation from the mean is <10% of
the mean value.
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
Percent of nitric oxide released based on a theoretical maximum release of (i) 1 mol of NO/mol of the oxynitrate test compounds (15a–b, 17a–b) and (ii) 3 mol of NO/mol
c
of glycerol trinitrate (18). The result is the mean value of three measurements (n = 3) where variation from the mean% value was 60.2%.
d
A solution of the test compound (2.4 mL of a 1.0 Â 10À2 mM solution in phosphate buffer at pH 7.4, was incubated at 37 °C for 1.5 h.
e
A solution of the test compound (2.4 mL of a 1.0 Â 10À2 mM solution in phosphate buffer at pH 7.4 which contained 5.0 mM
L-cysteine), was incubated at 37 °C for 1.5 h.
f
Inhibitory activity in a carrageenan-induced rat paw edema assay. The results are expressed as the ED50 value (mg/kg) at 3 h after oral administration of the test
compound.
g
A 9.0% inhibition of inflammation was observed using a 100 mg/kg oral dose.
Inactive at a 100 mg/kg oral dose.
Data acquired using ovine COX-2 (Catalog No. 56101, Cayman Chemical Inc.).
h
i
major determinant of COX-2 inhibitory potency since compounds
15a and 15b, and 17a and 17b, show similar potencies. Compounds
17a–b having a 1,2,3,6-tetrahydropyridyl ring are moderately
more potent COX-2 inhibitors compared to the corresponding
piperidyl compounds 15a–b. This difference in potency may be
due to the fact that the 1,2,3,6-tetrahydropyridyl ring is attached
via a sp2 hybridized carbon while the piperidyl ring is attached
via a sp3 hybridized carbon. The most potent COX-2 inhibitor
1,2,3,6-tetrahydropyridyl compound 17a having a H2NSO2 moiety
is about equipotent with aspirin but is less potent than celecoxib
and ibuprofen.
The percent NO released from the hybrid prodrugs 15a–b and
17a–b upon incubation in phosphate-buffered-saline (PBS at pH
7.4), varied over a narrow range (2.4–5.8%) which is indicative of
slow NO release26 (see data in Table 1). The percentage of NO re-
leased from the N-nitrooxybutyl compounds 15a–b and 17a–b
1,2,3,6-tetrahydropiperidyl (17a > 17b) moiety is present, (ii) with-
in the N-(4-nitrooxybutyl)piperidyl group of compounds, 15a
(ED50 = 132.4 mg/kg po) is a more potent AI agent than 15b which
showed a weak 9.0% inhibition of inflammation for a 100 mg/kg
oral dose, and (iii) within the N-(4-nitrooxybutyl)-1,2,3,6-
tetrahydropyridyl group of compounds, 17a exhibited AI activity
(ED50 = 118.4 mg/kg po) between that of aspirin (ED50
=
128.7 mg/kg po), and ibuprofen (ED50 = 67.4 mg/kg po). In contrast,
the sulfonamide compound 17b showed no inhibition of inflam-
mation at the maximal dose employed (100 mg/kg oral dose).
In conclusion, a new class of 1-(4-methane(amino)sulfonyl-
phenyl)-5-[1-(4-nitrooxybutyl)piperidin-4-yl]-3-trifluoromethyl-
1H-pyrazoles (15a–b) and 1-(4-methane(amino)sulfonylphenyl)-
5-[1-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-yl]-3-trifluoro-
methyl-1H-pyrazoles (17a–b) was synthesized29 for evaluation as
COX-1/COX-2 isozyme inhibitors,30 NO donors,31 and as anti-
inflammatory agents.32 The structure–activity data acquired indi-
cate that (i) compounds 15a–b and 17a–b exhibit weak in vitro
COX-2 inhibitory activity, (ii) compounds having a MeSO2 COX-2
pharmacophore show superior AI activity compared to the corre-
sponding H2NSO2 compounds, (iii) both classes of prodrugs (15a–
b and 17a–b) are relatively stable in phosphate-buffered saline at
was higher in the presence of 5 mM
L-cysteine (3.1–8.4% range)
than in the absence of -cysteine. This latter observation is consis-
L
tent with reports that NO release from organic nitrates is facili-
tated by thiols.27,28
The oral AI activities exhibited by the piperidyl 15a–b, and
1,2,3,6-tetrahydropyridyl 17a–b, compounds were determined
using a carrageenan-induced rat foot paw edema model (see data
in Table 1). Structure–activity relationship data showed that the
relative AI potency profile (i) with respect to the COX-2 pharmaco-
phore moiety was SO2Me > SO2NH2 irrespective of whether a N-(4-
pH 7.4 where NO release is in the 2.4–5.8% range, (iv) L-cysteine
moderately enhances the release of NO (3.1–8.4% range), and (v)
modification of celecoxib by replacement of the H2NSO2 by MeSO2,
and the para-tolyl moiety by either a N-(4-nitrooxybutyl)piperid-
4yl (15a) or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyrid-4yl
nitrooxybutyl)piperidyl (15a
> 15b) or N-(4-nitrooxybutyl)-

1328
M. A. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1324–1329
General procedure for the synthesis of 1-(4-methane(amino)sulfonylphenyl)-5-
(piperidin-4-yl)-3-trifluoromethyl-1H-pyrazoles (11a–b): Palladium-on-charcoal
(17a) moiety furnishes compounds that exhibit AI activity between
that of the reference drugs aspirin and ibuprofen.
(1.13 g of 10% w/w) was added to
a
solution of one of 1-(4-
methane(amino)sulfonylphenyl)-5-(pyridin-4-yl)-3-trifluoromethyl-1H-pyrazole
(10a or 10b, 4.08 mmol) in acetic acid (50 mL). The resulting suspension was
then flushed with argon followed by three consecutive flushes with H2 gas
to remove any air or argon from the hydrogenation flask. The pressure in
the hydrogenation flask was maintained at 60 psi with H2 gas using a Parr
apparatus. After shaking for 20 h the H2 gas was released from the
hydrogenation flask and the reaction mixture was filtered through a celite pad
to remove any Pd/C catalyst. The filtrate was evaporated in vacuo to give a solid
product as the acetate salt that was treated with a saturated Na2CO3 solution
(100 mL) prior to extraction with ethyl acetate (3 Â 100 mL). The combined
organic fractions were washed successively with water and brine, and the
organic fraction was dried (MgSO4). Filtration and then removal of the solvent
from the organic fraction in vacuo afforded the crude product which was
recrystallized with acetone–hexanes to furnish the respective title compound
11a or 11b. Some physical and spectroscopic data for 11a–b are listed below.
Acknowledgment
We are grateful to the Canadian Institutes of Health Research
(CIHR) (MOP-14712) for financial support of this research.
References and notes
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(11a):
This compound was obtained as white crystals in 77% yield; mp 174–176 °C;
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6. Scheen, A. J. Rev. Med. Liege 2004, 59, 565.
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;
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10. Ranatunge, R. R.; Augustyniak, M.; Bandarage, U. K.; Earl, R. A.; Ellis, J. L.;
Garvey, D. S.; Janero, D. R.; Letts, L. G.; Martino, A. M.; Murty, M. G.; Richardson,
S. K.; Schroeder, J. D.; Shumway, M. J.; Tam, S. W.; Trocha, M.; Young, D. V. J.
Med. Chem. 2004, 47, 2180.
11. Abdellatif, K. R. A.; Chowdhury, M. A.; Dong, Y.; Das, D.; Yu, G.; Velázquez, C. A.;
Suresh, M. R.; Knaus, E. E. Bioorg. Med. Chem. Lett. 2009, 19, 3014.
12. Chiroli, V.; Benedini, F.; Ongini, E.; Del Soldato, P. Eur. J. Med. Chem. 2003, 38,
441.
13. Corazzi, T.; Leone, M.; Maucci, R.; Corazzi, L.; Gresele, P. J. Pharmacol. Exp. Ther.
2005, 315, 1331.
14. Yang, C.-F.; Zhang, Y.-Y.; Yang, B.; Li, P.-F.; Zhuang, D.-Y. Zhongguo Xinyao Zazhi
2004, 13, 818.
15. Downing, J. E. G.; Madden, J. C.; Ingram, M. J.; Rostron, C. Biochem. Biophys. Res.
Commun. 2005, 334, 646.
16. Proszenyák, Á.; Ágai, B.; Hegedus, L.; Faigl, F. Appl. Catal., A 2004, 269, 249.
(dddd, J = 12.2, 12.2, 12.2, 3.7 Hz, 2H, piperidyl H-3ax, H-5ax), 1.81 (br d,
J = 12.2 Hz, 2H, piperidyl H-3 equiv, H-5 equiv), 2.61 (ddd, J = 12.2, 12.2, 1.9 Hz,
2H, piperidyl H-2ax, H-6ax), 2.80 (dddd, J3ax,4ax = 12.2, J4ax,5ax = 12.2,
J3equiv,4ax = 3.7, J4ax,5equiv = 3.7 Hz, 1H, piperidyl H-4ax), 3.12 (s, 3H, SO2CH3),
3.13 (br d, J = 12.2 Hz, 2H, piperidyl H-2 equiv, H-6 equiv), 6.55 (s, 1H, pyrazole
H-4), 7.66 (d, J = 8.6 Hz, 2H, phenyl H-2, H-6), 8.11 (d, J = 8.6 Hz, 2H, phenyl H-3,
H-5). Anal. Calcd for C16H18F3N3O2S: C, 51.47; H, 4.86; N, 11.25; S, 8.59. Found: C,
51.24; H, 4.82; N, 10.89; S, 8.46.
1-(4-Aminosulfonylphenyl)-5-(piperidin-4-yl)-3-trifluoromethyl-1H-pyrazole
(11b): This compound was obtained as white crystals in 70% yield; mp 205–
206 °C; IR (film) 3320 (br NH), 2943 (C–H aromatic), 2858 (C–H aliphatic), 1350,
1166 (SO2) cmÀ1 1H NMR (DMSO-d6) d 1.45 (dddd, J = 12.2, 12.2, 12.2, 3.7 Hz,
;
2H, piperidyl H-3ax, H-5ax), 1.66 (br d, J = 12.2 Hz, 2H, piperidyl H-3 equiv, H-
5 equiv), 2.39 (ddd, J = 12.2, 12.2, 1.9 Hz 2H, piperidyl H-2ax, H-6ax), 2.78 (dddd,
J3ax,4ax = 12.2, J4ax,5ax = 12.2, J3equiv,4ax = 3.7, J4ax,5equiv = 3.7 Hz, 1H, piperidyl H-
4ax), 2.90 (br d, J = 12.2 Hz, 2H, piperidyl H-2 equiv, H-6 equiv), 3.35 (br s, 1H,
NH that exchanges with D2O), 6.87 (s, 1H, pyrazole H-4), 7.40 (br s, 2H, SO2NH2
that exchanges with D2O), 7.77 (d, J = 8.6 Hz, 2H, phenyl H-2, H-6), 7.99 (d,
J = 8.6 Hz, 2H, phenyl H-3, H-5). Anal. Calcd for C15H17F3N4O2SÁ1.3H2O: C, 47.36;
H, 4.68. Found: C, 47.57; H, 4.72.
}
17. Toscano, J. P.; Pavlos, C. M.; Boppana, K. International PCT Patent, WO 2005/
074598 A2, Issued August 18, 2005.
1-(4-Methanesulfonylphenyl)-5-(1-nitrosopiperidin-4-yl)-3-trifluoromethyl-1H-
pyrazole (12): The piperidine compound 11a (200 mg, 0.53 mmol) was added to
a solution of NaOMe (65 mg of 95% purity, 0.53 mmol) in THF (10 mL) with
stirring at À65 °C. This mixture was purged with argon for 5 min, and the
reaction was allowed to proceed under an atmosphere of nitric oxide gas (40 psi
internal pressure) with stirring at À65 °C for 1 h. The reaction mixture was
allowed to warm to 25 °C and filtered to remove some inorganic materials. The
solvent was removed from the filtrate to furnish product 12 in 67% yield. In a
second reaction, nitric oxide gas was bubbled into a solution of 11a using a
protic solvent system consisting of NaOMe in MeOH (25% w/v) and MeCN at
25 °C which afforded 12 in 98% yield; mp 202–203 °C; IR (film) 3028 (C–H
18. Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J. S.; Collins, P. W.; Docter,
S.; Graneto, M. J.; Lee, L. F.; Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier,
D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.;
Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.;
Isakson, P. C. J. Med. Chem. 1997, 40, 1347.
19. Rao, P. N. P.; Amini, M.; Li, H.; Habeeb, A. G.; Knaus, E. E. J. Med. Chem. 2003, 46,
4872. and references cited therein.
20. Abdellatif, K. R. A.; Dong, Y.; Chen, Q.-H.; Chowdhury, M. A.; Knaus, E. E. Bioorg.
Med. Chem. 2007, 15, 6796.
21. Abdellatif, K. R. A.; Chowdhury, M. A.; Dong, Y.; Chen, Q.-H.; Knaus, E. E. Bioorg.
Med. Chem. 2008, 16, 3302.
22. Velázquez, C. A.; Praveen Rao, P. N.; Citro, M. L.; Keefer, L. K.; Knaus, E. E. Bioorg.
Med. Chem. 2007, 15, 4767.
23. Velázquez, C. A.; Chen, Q.-H.; Citro, M. L.; Keefer, L. K.; Knaus, E. E. J. Med. Chem.
2008, 51, 1954.
24. Abdellatif, K. R. A.; Chowdhury, M. A.; Dong, Y.; Knaus, E. E. Bioorg. Med. Chem.
2008, 16, 6528.
aromatic), 2931 (C–H aliphatic), 1317, 1150 (SO2) cmÀ1 1H NMR (CDCl3) d 1.55
;
and 1.90 (dddd, J = 12.2, 12.2, 12.2, 3.7 Hz, 1H each, piperidyl H-3ax and H-5ax),
1.94 and 2.10 (br d, J = 12.2 Hz, 1H each, piperidyl H-3 equiv and H-5 equiv),
2.51 and 3.68 (ddd, J = 12.8, 12.8, 3.1 Hz, 1H each, piperidyl H-2ax and H-6ax),
3.11 (dddd, J = 12.2, 12.2, 3.7, 3.7 Hz, 1H, piperidyl H-4ax), 3.12 (s, 3H, SO2CH3),
4.90 and 5.13 (dd, J = 12.1, 2.4 Hz, 1H each, piperidyl H-2 equiv and H-6 equiv),
6.54 (s, 1H, pyrazole H-4), 7.70 (dd, J = 8.6, 2.5 Hz, 2H, phenyl H-2, H-6), 8.14 (dd,
J = 8.6, 2.5 Hz, 2H, phenyl H-3, H-5). Anal. Calcd for C16H17F3N4O3S: C, 47.76; H,
4.26. Found: C, 47.92; H, 4.25.
25. Garvey, D. S.; Letts, L. G.; Earl, R. A.; Ezawa, M.; Fang, X.; Gaston, R. D.;
Khanapure, S. P.; Lin, C.-E.; Ranatunge, R. R.; Stevenson, C. A.; Wey, S.-J. U.S. Pat.
Appl. Publ. 2006, US 2006189603 A1.
General procedure for the synthesis of 1-(4-methane(amino)sulfonylphenyl)-5-[1-
(4-nitrooxybutyl)piperidin-4-yl]-3-trifluoromethyl-1H-pyrazoles (15a–b) and 1-
(4-methane(amino)sulfonylphenyl)-5-[1-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyri
din-4-yl]-3-trifluoromethyl-1H-pyrazoles (17a–b): Cesium carbonate (0.22 g,
0.67 mmol) and 4-nitrooxybutyl bromide (14, 0.27 g, 1.34 mmol) were added
to a solution of the piperidine 11a or 11b, or 1,2,3,6-tetrahydropyridine 16a or
16b, compound (1.34 mmol) in dry DMF (4 mL). The reaction mixture was
stirred at 25 °C for 24 h, and water (10 mL) was added prior to extraction with
EtOAc (3 Â 20 mL). The combined organic fractions were washed successively
with water and brine, and the organic fraction was dried (MgSO4). Filtration and
removal of the solvent from the organic fraction in vacuo afforded the crude
product which was purified by silica gel column chromatography using
hexanes/EtOAc (1:3, v/v) as eluent to furnish the respective title compound
15a–b or 17a–b. The low isolated yields of 15a–b and 17b is attributed to
product decomposition during column purification. Some physical and
spectroscopic data for 15a–b and 17a–b are listed below.
26. Saavedra, J. E.; Shami, P. J.; Wang, L. Y.; Davies, K. M.; Booth, M. N.; Citro, M. L.;
Keefer, L. K. J. Med. Chem. 2000, 43, 261.
27. Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk, A. J. Chem. Rev.
2002, 102, 1091. and references cited therein.
28. Shan, R.; Velázquez, C. A.; Knaus, E. E. J. Med. Chem. 2004, 47, 254.
29. Experimental procedures and spectral data for compounds 11, 12, 15 and 17.
General. Melting points were determined on
a Thomas–Hoover capillary
apparatus and are uncorrected. Unless otherwise noted, infrared (IR) spectra
were recorded as films on NaCl plates using a Nicolet 550 Series II Magna FT-IR
spectrometer. 1H NMR and 13C NMR spectra were measured on a Bruker AM-
300 spectrometer. Microanalyses (Micro Analytical Service Laboratory,
Department of Chemistry, University of Alberta) were performed for C and H
unless otherwise stated, and were within 0.4% of theoretical values for all
elements listed. Silica gel column chromatography was performed using Merck
Silica Gel 60 ASTM (70–230 mesh). 1-(4-Methane(amino)sulfonylphenyl)-5-
(pyridine-4-yl)-3-trifluoromethyl-1H-pyrazoles (10a and 10b) and 1-(4-
methane(amino)sulfonylphenyl)-5-(1,2,3,6-tetrahydropyridin-4-yl]-3-trifluoro-
methyl-1H-pyrazole (16a and 16b) were prepared according to our previously
reported procedure (Chowdhury, M. A.; Abdellatif, K. R. A.; Dong, Y.; Knaus, E.
E. Bioorg. Med. Chem. 2008, 16, 8882). All other reagents, purchased from the
Aldrich Chemical Company (Milwaukee, WI), were used without further
purification. The in vivo anti-inflammatory assay was carried out using a
protocol approved by the Health Sciences Animal Welfare Committee at the
University of Alberta.
1-(4-Methanesulfonylphenyl)-5-[1-(4-nitrooxybutyl)piperidin-4-yl]-3-trifluorome
thyl-1H-pyrazole (15a): The product was obtained as a colorless gum in 23%
yield; IR (film) 2957 (C–H aromatic), 2850 (C–H aliphatic), 1695, 1627, 1280
(ONO2), 1319, 1150 (SO2) cmÀ1 1H NMR (CDCl3) d 1.52–1.70 (m, 2H, piperidyl
;
H-3ax, H-5ax), 1.70–1.90 (m, 6H, piperidyl H-3 equiv, H-5 equiv,
CH2CH2CH2ONO2), 2.65–2.83 (m, 2H, piperidyl H-2ax, H-6ax), 2.87 (dddd,
J = 12.2, 12.2, 3.7, 3.7 Hz, 1H, piperidyl H-4ax), 3.14 (s, 3H, SO2Me), 4.14 (t,
J = 5.5 Hz, 2H, NCH2), 4.07–4.35 (m, 2H, piperidyl H-2 equiv, H-6 equiv), 4.50 (t,

M. A. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1324–1329
1329
J = 6.1 Hz, 2H, CH2ONO2), 6.55 (s, 1H, pyrazole H-4), 7.68 (d, J = 8.6 Hz, 2H,
phenyl H-2, H-6), 8.15 (d, J = 8.6 Hz, 2H, phenyl H-3, H-5). Anal. Calcd for
C20H25F3N4O5SÁ1/6H2O: C, 48.67; H, 5.17. Found: C, 48.28; H, 4.98.
gum in 27% yield; IR (film) 3232, 3104 (br NH2), 2930 (C–H aromatic), 2855 (C–H
aliphatic), 1684, 1627, 1281 (ONO2), 1325, 1161 (SO2) cmÀ1 1H NMR (DMSO-d6)
;
d 1.58–1.78 (m, 4H, CH2CH2CH2ONO2), 2.18–2.22 (m, 2H, tetrahydropyridyl H-
3), 3.44-3.47 (m, 2H, tetrahydropyridyl H-2), 3.90-3.92 (m, 2H,
tetrahydropyridyl H-6), 4.03 (t, J = 6.1 Hz, 2H, NCH2), 4.53 (t, J = 6.1 Hz, 2H,
CH2ONO2), 5.85 (br s, 1H, tetrahydropyridyl H-5), 7.05 (s, 1H, pyrazole H-4), 7.55
(s, 2H, SO2NH2 that exchanges with D2O), 7.73 (d, J = 8.6 Hz, 2H, phenyl H-2, H-
6), 7.96 (d, J = 8.6 Hz, 2H, phenyl H-3, H-5). Anal. Calcd for C19H22F3N5O5SÁ1/
4H2O: C, 46.19; H, 4.59. Found: C, 46.02; H, 4.91.
1-(4-Aminosulfonylphenyl)-5-[1-(4-nitrooxybutyl)piperidin-4-yl]-3-trifluorometh
yl-1H-pyrazole (15b): The product was obtained as a colorless gum in 11% yield;
IR (film) 3242 (br NH2), 2928 (C–H aromatic), 2850 (C–H aliphatic), 1682, 1624,
1275 (ONO2), 1320, 1160 (SO2) cmÀ1 1H NMR (CDCl3) d 1.55–1.71 (m, 2H,
;
piperidyl H-3ax, H-5ax), 1.71-1.90 (m, 6H, piperidyl H-3 equiv, H-5 equiv,
CH2CH2CH2ONO2), 2.65–2.87 (m, 3H, piperidyl H-2ax, H-4ax, H-6ax), 4.14 (t,
J = 5.5 Hz, 2H, NCH2), 4.05–4.35 (m, 2H, piperidyl H-2 equiv, H-6 equiv), 4.50 (t,
J = 6.1 Hz, 2H, CH2ONO2), 5.04 (br s, 2H, SO2NH2 that exchanges with D2O), 6.54
(s, 1H, pyrazole H-4), 7.62 (d, J = 8.6 Hz, 2H, phenyl H-2, H-6), 8.12 (d, J = 8.6 Hz,
2H, phenyl H-3, H-5). Anal. Calcd for C19H24F3N5O5S: C, 46.43; H, 4.92. Found: C,
46.20; H, 4.76.
30. Cyclooxygenase inhibition assays: The ability of the test compounds listed in
Table 1 to inhibit ovine COX-1 and human recombinant COX-2 (IC50 value, lM)
was determined using an enzyme immuno assay (EIA) kit (Catalog No. 560131,
Cayman Chemical, Ann Arbor, MI, USA) according to our previously reported
method (Rao, P. N. P.; Amini, M.; Li, H.; Habeeb, A.; Knaus, E. E. J. Med. Chem.
2003, 46, 4872).
1-(4-Methanesulfonylphenyl)-5-[1-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-
yl]-3-trifluoromethyl-1H-pyrazole (17a): The product was obtained as a colorless
gum in 47% yield; IR (film) 2930 (C–H aromatic), 2850 (C–H aliphatic), 1699,
31. Nitric oxide release assays: In vitro nitric oxide release, upon incubation of the
test compound at 37 °C for 1.5 h with 2.4 mL of a 1.0 Â 10À2 mM solution in
phosphate buffer at pH 7.4 was determined by quantification of nitrite
produced by the reaction of nitric oxide with oxygen and water using the
Griess reaction. Nitric oxide release data were acquired for test compounds
(15a–b, 17a–b) using the reported procedures (Velázquez, C.; Vo, D.; Knaus, E.
E. Drug Dev. Res. 2003, 60, 204).
32. In vivo anti-inflammatory assay: The test compounds 15a–b, 17a–b, and the
reference drugs celecoxib, aspirin, and ibuprofen were evaluated using the
in vivo carrageenan-induced rat foot paw edema model reported previously
(Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544).
1627, 1280 (ONO2), 1325, 1160 (SO2) cmÀ1 1H NMR (DMSO-d6) d 1.57–1.77 (m,
;
4H, CH2CH2CH2ONO2), 2.22–2.24 (m, 2H, tetrahydropyridyl H-3), 3.30 (s, 3H,
SO2Me), 3.40–3.55 (m, 2H, tetrahydropyridyl H-2), 3.91–3.93 (m, 2H,
tetrahydropyridyl H-6), 4.04 (t, J = 6.1 Hz, 2H, NCH2), 4.54 (t, J = 6.1 Hz, 2H,
CH2ONO2), 5.84–5.86 (m, 1H, tetrahydropyridyl H-5), 7.07 (s, 1H, pyrazole H-4),
7.81 (d, J = 8.6 Hz, 2H, phenyl H-2, H-6), 8.08 (d, J = 8.6 Hz, 2H, phenyl H-3, H-5).
Anal. Calcd for C20H23F3N4O5SÁ2H2O: C, 45.80; H, 5.19. Found: C, 46.09; H, 4.90.
1-(4-Aminosulfonylphenyl)-5-[1-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-yl
]-3-trifluoromethyl-1H-pyrazole (17b): The product was obtained as a colorless